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Numéro de publicationUS8889348 B2
Type de publicationOctroi
Numéro de demandeUS 12/308,091
Numéro PCTPCT/US2007/013559
Date de publication18 nov. 2014
Date de dépôt7 juin 2007
Date de priorité7 juin 2006
Autre référence de publicationCN101495656A, CN101495656B, US20090298072, US20150037788, US20170096704, WO2007146158A1
Numéro de publication12308091, 308091, PCT/2007/13559, PCT/US/2007/013559, PCT/US/2007/13559, PCT/US/7/013559, PCT/US/7/13559, PCT/US2007/013559, PCT/US2007/13559, PCT/US2007013559, PCT/US200713559, PCT/US7/013559, PCT/US7/13559, PCT/US7013559, PCT/US713559, US 8889348 B2, US 8889348B2, US-B2-8889348, US8889348 B2, US8889348B2
InventeursJingyue Ju
Cessionnaire d'origineThe Trustees Of Columbia University In The City Of New York
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes: USPTO, Cession USPTO, Espacenet
DNA sequencing by nanopore using modified nucleotides
US 8889348 B2
Résumé
This invention provides a process for sequencing single-stranded DNA by employing a nanopore and modified nucleotides.
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Revendications(19)
What is claimed is:
1. A method for determining the nucleotide sequence of a single-stranded DNA comprising the steps of:
(a) synthesizing a precursor of the single-stranded DNA as part of a double-stranded DNA using a single-stranded DNA template, a primer, a DNA polymerase, and all four nucleotides present in DNA, wherein each A or each G residue, but not both, comprise a first label bound to its respective base, and each C or each T residue, but not both, comprise a second label bound to its respective base;
(b) denaturing the double-stranded DNA obtained in (a);
(c) isolating the single-stranded DNA to be sequenced comprising the nucleotides containing the first label and the second label from the denatured double-stranded DNA;
(d) reacting the single-stranded DNA isolated in (c) with first modifying groups which form bonds with the first labels and second modifying groups which form bonds with the second labels, so as to obtain the single-stranded DNA to be sequenced wherein each labeled nucleotide is conjugated to a modifying group;
(e) passing the single-stranded DNA to be sequenced through a pore of suitable diameter by applying and electric field to the DNA:
(f) determining an electronic signature for each nucleotide of the single-stranded DNA which passes through the pore; and
(g) comparing each electronic signature determined in (f) with electronic signatures corresponding to each of A, G, C, and T or corresponding to the bases conjugated to the modifying group, so as to determine the identity of each such nucleotide,
thereby determining the nucleotide sequence of the single-stranded DNA.
2. The method of claim 1, wherein the pore has a diameter of from about 1 nm to about 5 nm.
3. The method of claim 1, wherein the pore has a diameter of from about 1 nm to about 3 nm.
4. The method of claim 1, wherein the pore has a diameter of about 1 nm, 2 nm, 3 nm, 4 nm or 5 nm.
5. The method of claim 1, wherein each A and each T nucleotide comprises a modifying group bound to its labeled base.
6. The method of claim 1, wherein each G and each C nucleotide comprises a modifying group bound to its labeled base.
7. The method of claim 1, wherein each A and each C residue or each G and each T residue comprises a modifying group bound to its respective base.
8. The method of claim 1, wherein 5′-end of the primer comprises a biotin moiety.
9. The method of claim 1, wherein the first label is an amino group and the second label is an azido group.
10. The method of claim 1, wherein the first label is an azido group and the second label is an amino group.
11. A method for determining the nucleotide sequence of a single-stranded DNA comprising the steps of:
(a) passing the single-stranded DNA through a nanopore of a suitable diameter by applying an electric field, wherein each A or each G residue, but not both, comprises a modifying group bound to a label added to its respective base, and each C or each T residue, but not both, comprises a modifying group bound to a label added to its respective base, and each type of nucleotide in the single-stranded DNA has a characteristic electronic signature which is distinguishable from the electronic signature of all other types of nucleotides in the single-stranded DNA;
(b) determining the electronic signature for each nucleotide within the single-stranded DNA which passes through the nanopore
(c) comparing each electronic signature determined in step (b) with the electronic signature characteristic of each type of nucleotide so as to determine the identity of each such nucleotide that passes through the nanopore;
thereby determining the nucleotide sequence of the single-stranded DNA.
12. The method of claim 11, wherein the nanopore has a diameter of from about 1 nm to about 5 nm.
13. The method of claim 11, wherein the single-stranded DNA whose sequence is to be determined is obtained by:
(a) synthesizing the single-stranded DNA as part of a double-stranded DNA using a single-stranded DNA template, a primer, a DNA polymerase, and nucleotides, wherein each A or each G residue, but not both, comprises a modifying group bound to a label added to its respective, and each C or each T residue, but not both, comprises a modifying group bound to a label added to its respective base, wherein all of the A or G nucleotides, and all of the C or the T nucleotides within the single-stranded DNA comprise an identical modifying group bound to the label added to their respective bases and each type of nucleotide has a characteristic electronic signature which is distinguishable from the electronic signature of each other type of nucleotide in the DNA;
(b) denaturing the double-stranded DNA obtained in step (a); and
(c) isolating the single-stranded DNA to be sequenced comprising modified nucleotides from the denatured double-stranded DNA.
14. The method of claim 11, wherein the single-stranded DNA whose sequence is to be determined is obtained by:
(a) synthesizing a precursor of the single-stranded DNA as part of a double-stranded DNA using a single-stranded DNA template, a primer, a DNA polymerase, and all four nucleotides present in DNA, wherein each A or each G residue, but not both, comprise a first label bound to its respective base, and each C or each T residue, but not both, comprise a second label bound to its respective base;
(b) denaturing the double-stranded DNA obtained in (a);
(c) isolating the single-stranded DNA to be sequenced comprising the first label and the second label-containing nucleotides from the denatured double-stranded DNA; and
(d) reacting the single-stranded DNA isolated in (c) with first modifying groups which form bonds with the first labels and second modifying groups which form bonds with the second labels,
so as to obtain the single-stranded DNA to be sequenced.
15. The method of claim 14, wherein the first label is an amino group and the second label is an azido group.
16. The method of claim 14, wherein the first label is an azido group and the second label is an amino group.
17. The method of claim 11, wherein each A nucleotide of said single stranded DNA comprises a modifying group bound to its labeled base.
18. The method of claim 11, wherein each T nucleotide of said single stranded DNA comprises a modifying group bound to its labeled base.
19. The method of claim 11, wherein A, G, C, and T nucleotides of said single-stranded DNA have an order of bulkiness that is T>A>G>C.
Description

This application is a §371 national stage of PCT International Application No. PCT/US2007/013559, filed Jun. 7, 2007, and claims the benefit of U.S. Provisional Application No. 60/811,912, filed Jun. 7, 2006, the contents of all of which are hereby incorporated by reference into this application.

The invention disclosed herein was made with government support under grant no. 1R21HG003718-01 from the National Human Genome Research Institute. Accordingly, the U.S. Government has certain rights in this invention.

Throughout this application, various publications are referenced in parentheses by number. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

DNA sequencing is a fundamental technology for biology. Several analytical methods have been developed to detect DNA or RNA at the single molecule level using chemical or physical microscopic technologies [15, 16, 21 and 23]. In the past few years, the ion channel has been explored for detecting individual DNA or RNA strands, with nanopore being a candidate for high rate sequencing and analysis of DNA [9, 10, 4, 3 and 7].

In 1996, Kasianowicz et al. first demonstrated that the α-hemolysin channel, an exotoxin secreted by a bacterium, could be used to detect nucleic acids at the single molecule level [8]. The monomeric polypeptide self-assembles in a lipid bilayer membrane to form a heptameric pore, with a 2.6 nm-diameter vestibule and 1.5 nm-diameter limiting aperture (namely, the narrowest point of the pore) [1, 14 and 15]. In an aqueous ionic salt solution such as KCl, the pore formed by the α-hemolysin channel conducts a sufficiently strong and steady ionic current when an appropriate voltage is applied across the membrane. The limiting aperture of the nanopore allows linear single-stranded but not double-stranded nucleic acid molecules (diameter −2.0 nm) to pass through. The polyanionic nucleic acids are driven through the pore by the applied electric field, which blocks or reduces the ionic current that would be otherwise unimpeded. This process of passage generates an electronic signature (FIG. 1) [23 and 5]. A particular nucleic acid molecule, when entering and passing through the nanopore, will generate a characteristic signature that distinguishes it from others. The duration of the blockade is proportional to the length of the nucleic acid, and its signal strength is related to the steric and electronic properties of the nucleotides, namely the identity of the four bases (A, C, G and T).

A specific event diagram is constructed which is the plot of translocation time versus blockade current. This specific event diagram (also referred to as an electronic signature) is used to distinguish the lengths and the compositions of polynucleotides by single-channel recording techniques based on characteristic parameters such as translocation current, translocation duration, and their corresponding dispersions in the diagram [14].

Although the nanopore approach is known as a DNA detection method, this approach for base-to-base sequencing has not yet been achieved.

SUMMARY OF THE INVENTION

This invention provides a method for determining the nucleotide sequence of a single-stranded DNA comprising the steps of:

    • (a) passing the single-stranded DNA through a pore of suitable diameter by applying an electric field to the DNA, wherein at least each A or each G residue and at least each C, each T or each U residue comprises a modifying group bound to its respective base so that each type of nucleotide in the DNA has an electronic signature which is distinguishable from the electronic signature of each other type of nucleotide in the DNA;
    • (b) for each nucleotide of the DNA which passes through the pore, determining an electronic signature for such nucleotide; and
    • (c) comparing each electronic signature determined in step (b) with electronic signatures corresponding to each of A, G, C and T modified as per the nucleotides in the single-stranded DNA, so as to determine the identity of each such nucleotide,
      thereby determining the nucleotide sequence of the single-stranded DNA.

This invention also provides a method for determining the nucleotide sequence of a single-stranded RNA comprising the steps of:

    • (a) passing the single-stranded RNA through a pore of suitable diameter by applying an electric field to the RNA, wherein at least each A or each G residue and at least each C or each U residue comprises a modifying group bound to its respective base so that each type of nucleotide in the RNA has an electronic signature which is distinguishable from the electronic signature of each other type of nucleotide in the RNA;
    • (b) for each nucleotide of the RNA which passes through the pore, determining an electronic signature for such nucleotide; and
    • (c) comparing each electronic signature determined in step (b) with electronic signatures corresponding to each of A, G, C and U modified as per the nucleotides in the single-stranded RNA, so as to determine the identity of each such nucleotide,
      thereby determining the nucleotide sequence of the single-stranded RNA.

This invention also provides a nucleotide having an azido group covalently bound to its base.

This invention also provides a method for making a modified nucleotide comprising contacting the instant nucleotide with an alkyne-containing compound under conditions permitting reaction between the azido and the alkyne groups, thereby making the modified nucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. α-Hemolysin protein self-assembles in a lipid bilayer to form an ion channel and a nucleic acid stretch passes through it (left), with the corresponding electronic signatures, generated (right) [23 and 5].

FIG. 2. Structures of nucleotides dATP, dGTP, dCTP and dTTP.

FIG. 3. Nucleotide bulkiness in ascending order: (a) 5′-C60T-3′, (b) 5′-G*60T-3′, (c) 5′-A*60T-3′, and (d) 5′-T*60T-3′.

FIG. 4. Structures of dCTP and dGTP, and modified nucleotides (dATP-NH2 and dUTP-NH2).

FIG. 5. Modification of dATP-NH2 and dUTP-NH2.

FIG. 6. DNA-extension reaction using modified nucleotides (dATP-NHCOR1 and dUTP-NHCOR2) to generate a modified single-stranded DNA chain. (SEQ ID NOs. 1 and 2 for template and primer, respectively).

FIG. 7. Steps of verifying sequencing capacity via nanopore using various DNA sequences. (SEQ ID NOs. 3-6 for part (i), top to bottom, respectively; SEQ ID NO:7 for part (ii); SEQ ID NO:8 for part (iii); and SEQ ID NO:9 for part (iv)).

FIG. 8. Structures of unmodified nucleotides (dCTP and dGTP) and hook-labeled nucleotides (dATP-NH2 and dUTP-N3). The amino and the azido groups function as hooks to conjugate with bulky groups after the nucleotides are incorporated into the DNA strand.

FIG. 9. Synthesis of dUTP-N3.

FIG. 10. DNA-extension reaction using hook-labeled nucleotides (dATP-NH2 and dUTP-N3) to generate a modified single-stranded DNA chain, which will then react with large functional groups (R1 and R3) selectively for distinct detection by nanopore. (SEQ ID NOs. 1 and 2 for template and primer, respectively)

DETAILED DESCRIPTION OF THE INVENTION Terms

As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

A Adenine;
C Cytosine;
DNA Deoxyribonucleic acid;
G Guanine;
RNA Ribonucleic acid;
T Thymine; and
U Uracil.

“Electronic signature” of a nucleotide passing through a pore via application of an electronic field shall include, for example, the duration of the nucleotide's passage through the pore together with the observed amplitude of current during that passage. Electronic signatures can be visualized, for example, by a plot of current (e.g. pA) versus time. Electronic signature for a DNA is also envisioned and can be, for example, a plot of current (e.g. pA) versus time for the DNA to pass through the pore via application of an electric field.

“Nanopore” includes, for example, a structure comprising (a) a first and a second compartment separated by a physical barrier, which barrier has at least one pore with a diameter, for example, of from about 1 to 10 nm, and (b) a means for applying an electric field across the barrier so that a charged molecule such as DNA can pass from the first compartment through the pore to the second compartment. The nanopore ideally further comprises a means for measuring the electronic signature of a molecule passing through its barrier. The nanopore barrier may be synthetic or naturally occurring in part. Barriers can include, for example, lipid bilayers having therein α-hemolysin, oligomeric protein channels such as porins, and synthetic peptides and the like. Barriers can also include inorganic plates having one or more holes of a suitable size. Herein “nanopore”, “nanopore barrier” and the “pore” in the nanopore barrier are sometimes used equivalently.

“Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).

“Type” of nucleotide refers to A, G, C, T or U.

Embodiments of the Invention

This invention provides a method for determining the nucleotide sequence of a single-stranded DNA comprising the steps of:

    • (a) passing the single-stranded DNA through a pore of suitable diameter by applying an electric field to the DNA, wherein at least each A or each G residue and at least each C, each T or each U residue comprises a modifying group bound to its respective base so that each type of nucleotide in the DNA has an electronic signature which is distinguishable from the electronic signature of each other type of nucleotide in the DNA;
    • (b) for each nucleotide of the DNA which passes through the pore, determining an electronic signature for such nucleotide; and
    • (c) comparing each electronic signature determined in step (b) with electronic signatures corresponding to each of A, G, C and T modified as per the nucleotides in the single-stranded DNA, so as to determine the identity of each such nucleotide,
      thereby determining the nucleotide sequence of the single-stranded DNA.

In an embodiment of the instant method, the single-stranded DNA is obtained by (a) synthesizing double-stranded DNA using a single-stranded template, a DNA polymerase and nucleotides, wherein at least each A or each G residue and at least each C or each T residue comprises a modifying group bound to its respective base so that each type of nucleotide in the DNA has an electronic signature which is distinguishable from the electronic signature of each other type nucleotide in the DNA, and (b) removing from the resulting double-stranded DNA the single-stranded DNA containing modified nucleotides.

In another embodiment of the instant method, the single-stranded DNA is obtained by (a) synthesizing double-stranded DNA using a single-stranded template, a DNA polymerase and nucleotides, wherein at least each A, each G, each C, each U or each T residue comprises an azido group bound to its base, and at least each A, each G, each C, each U and each T comprises an amino group bound to its base, whereby the azido and amino groups do not reside on the same type of base, (b) removing from the resulting double-stranded DNA the single-stranded. DNA containing the azido and amino group-containing nucleotides and (c) reacting the resulting single-stranded DNA with a first modifying group which forms a bond with the azido group and a second modifying group which forms a bond with the amino group so as to obtain the single-stranded DNA.

This invention also provides a method for determining the nucleotide sequence of a single-stranded RNA comprising the steps of:

    • (a) passing the single-stranded RNA through a pore of suitable diameter by applying an electric field to the RNA, wherein at least each A or each G residue and at least each C or each U residue comprises a modifying group bound to its respective base so that each type of nucleotide in the RNA has an electronic signature which is distinguishable from the electronic signature of each other type of nucleotide in the RNA;
    • (b) for each nucleotide of the RNA which passes through the pore, determining an electronic signature for such nucleotide; and
    • (c) comparing each electronic signature determined in step (b) with electronic signatures corresponding to each of A, G, C and U modified as per the nucleotides in the single-stranded RNA, so as to determine the identity of each such nucleotide,
      thereby determining the nucleotide sequence of the single-stranded RNA.

In an embodiment of the instant method, the single-stranded RNA is obtained by (a) synthesizing double-stranded RNA using a single-stranded template, an RNA polymerase and nucleotides, wherein at least each A, each G, each C or each U residue comprises an azido group bound to its base, and at least each A, each G, each C and each U comprises an amino group bound to its base, whereby the azido and amino groups do not reside on the same type of base, (b) removing from the resulting double-stranded RNA the single-stranded RNA containing the azido and amino group-containing nucleotides and (c) reacting the resulting single-stranded RNA with a first modifying group which forms a bond with the azido group and a second modifying group which forms a bond with the amino group so as to obtain the single-stranded RNA.

In another embodiment of the instant method, the single-stranded RNA is obtained by (a) synthesizing double-stranded RNA using a single-stranded template, an RNA polymerase and nucleotides, wherein at least each A or each G residue and at least each C or each U residue comprises a modifying group bound to its respective base so that each type of nucleotide in the RNA has an electronic signature which is distinguishable from the electronic signature of each other type nucleotide in the RNA, and (b) removing from the resulting double-stranded RNA the single-stranded RNA containing modified nucleotides.

In one embodiment of the instant methods, the pore has a diameter of from about 1 nm to about 5 nm. In a further embodiment of the instant methods, the pore has a diameter of from about 1 nm to about 3 nm. In embodiments of the instant methods, the pore has a diameter of about 1 nm, 2 nm, 3 nm, 4 nm or 5 nm. In further embodiments, the pore is, for example, about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 nm in diameter.

In one embodiment, a single pore is employed. In another embodiment, multiple pores are employed.

Nanopore devices are known in the art. See, for example, references [24] through [34]. Nanopores and methods employing them are disclosed in U.S. Pat. No. 7,005,264 B2 and U.S. Pat. No. 6,617,113 which are, hereby incorporated by reference in their entirety.

In one embodiment of the instant methods, each A and each T or each U residue comprises a modifying group; each A and each U residue comprises a modifying group; and/or each G and each C residue comprises a modifying group.

Moieties used to modify nucleotides can differ in size and/or charge, so long as each type of nucleotide in a nucleic acid whose sequence is being determined by the instant methods has an electronic signature which differs from each other type.

DNA polymerases which can be used in the instant invention include, for example E. Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase™, Taq DNA polymerase and 9° N polymerase (exo-) A485L/Y409V.RNA polymerases which can be used in the instant invention include, for example, Bacteriophage SP6, T7 and T3 RNA polymerases.

This invention also provides a nucleotide having an azido group covalently bound to its base. In one embodiment, the nucleotide is dUTP and the azido group is bound to the base at the 5-position. In one embodiment, the nucleotide is DATP and the azido group is bound to the base at the 8-position. In another embodiment, the nucleotide is dGTP and the azido group is bound to the base at the 8-position. The azido and amino groups can also be any other groups which permit binding of a unique moiety to each type of nucleotide.

This invention also provides a method for making a modified nucleotide comprising contacting the instant nucleotide with an alkyne-containing compound under conditions permitting reaction between the azido and the alkyne groups, thereby making the modified nucleotide.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

The structures of the four nucleotides are shown in FIG. 2. A and G are purines, while C and T are pyrimidines. The overall molecular sizes of A and G are very similar, while the sizes of C and T are similar. Thus, nanopore has been shown to be able to differentiate between purines and pyrimidines [1 and 14], but not to be able to distinguish between individual purinee, A and G, or between individual pyrimidines, C and T.

Disclosed here is the design of modified nucleotides to enhance discrimination of each nucleotide by modifying A and T. Since A and G are bulky purines similar in size, they will generate similar blocking current signatures (also called electronic signatures) in the nanopore. Likewise C and T, both pyrimidines, will generate similar signatures. The site selected for modification is on the 7-position of A and the 5-position of T nucleotide molecules. The 7-position of A and the 5-position of T have been shown to be chemically modified with bulky groups while not affecting basic DNA properties, such as forming the double-stranded DNA structure and being able to carry out polymerase reactions [2, 13 and 17]. These modifications will enlarge the discrimination of the bases by nanopore due to the increased size differences between the four nucleotides (A, G, C and T). In addition, the DNA translocation rate through the nanopore is expected to slow down due to the bulkiness of the modified nucleotides. Thus, achieving the accuracy and reliability required for the base-to-base sequencing is envisioned. The overall analytical parameters in the nanopore sequencing, such as concentration of the polynucleotide, magnitude of applied voltage, temperature and pH value of the solution, are optimized in order to get the most accurate and reliable results for the detection and analysis of the DNA chain.

Use of Synthetic DNA Carrying Bulky Groups for Detection by Nanopore

In order to investigate the effect of nucleotide bulkiness on electronic blockade signals generated by the nanopore, various polynucleotides are synthesized with different bulky groups attached to the base of the nucleotide by a DNA synthesizer. Initially, regular C's and G's are used to synthesize a series of polynucleotides (FIGS. 3 a and 3 b). In addition, a series of polynucleotides using modified A's (6-amino-hexylamino attached to the 8-position of the base) and modified T's (BIOTIN attached to the 5-position of the base) (FIGS. 3 c and 3 d), which increase the bulkiness of the nucleotides, are synthesized. The order of the bulkiness of the nucleotides in FIG. 3 is as follows: T*>A*>G>C. These polynucleotides are then passed through the nanopore to identify the relationship between the bulky groups attached to the base and the difference in electronic blockade signal between the different bases.

Attachment of Bulky Groups to Nucleotides for Nanopore Detection

(1) Design and Synthesis of Modified Nucleotides (dATP-NHCOR1 and dUTP-NHCOR2).

Synthesized dATP-NH2 and dUTP-NH2 are used as starting materials for further nucleotide modification while unmodified dCTP and dGTP are used directly (FIG. 4). The routes of nucleotide modification are shown in FIG. 5. The commercially available carboxylic acids 1˜10 will be converted into the corresponding N-hydroxysuccinimidyl (NHS) esters conveniently using N-hydroxysuccinimide and DCC [20 and 22]. Then the nucleotides for modification (dATP-NH2 and dUTP-NH2) will be connected with the modification groups R1 and R2 respectively in DMF and NaHCO3/Na2CO3 buffer solution [13 and 17]. After modification, the order of nucleotide bulkiness will be: A*>U*>G>C, as purines (A and G) are larger than pyrimidines (C and U) and in general the modification group R1 is larger than R2.

(2) DNA-Extension Reaction Using Modified Nucleotides (dATP-NHCOR1 and dUTP-NHCOR2).

The modified DATP and dUTP, and the unmodified dCTP and dGTP, are then be used in a polymerase reaction to generate single-stranded DNA. As shown in FIG. 6, after the polymerase reaction, the single-stranded DNA chain is obtained after being denatured from the template chain, which is composed of the modified DATP and dUTP as well as unmodified dCTP and dGTP. The 5′-end of the primer chain is modified on the base by a biotin moiety to isolate only DNA product that has incorporated the modified nucleotides. These modified single-stranded chains are then used in the nanopore by single-channel recording techniques for sequencing sensitivity and accuracy evaluation.

DNA-Sequencing Study by Nanopore

To validate nanopore's ability to distinguish the four different nucleotides in DNA, a series of tests are conducted as shown in FIG. 7. First, a polynucleotide stretch composed of only 50 identical nucleotides (i) is prepared by polymerase reaction as described above. Each DNA sequence is expected to generate different electronic blockade signatures due to the larger size difference of the nucleotides. The modification effects of R1 and R2 for A and T can be compared for preliminary optimization. Next, a polynucleotide stretch composed of 30 modified Ala and 30 modified T's (ii) is prepared and then tested in nanopore to demonstrate that the electronic blockade signatures differ in magnitude between A and T and are easily distinguishable.

Based on the signatures generated, the candidates for R1 and R2 groups are selected to achieve the best discrimination in signal. Third, a shorter polynucleotide stretch composed of 10 A's, 10 C's, 10 G's and 10 T's (iii) are prepared and tested in nanopore for further confirmation on the electronic blockade signatures (also called electronic signatures). Finally, a polynucleotide stretch composed of three consecutive A-C-G-T sequence (iv) is prepared and tested in nanopore. The detailed sequencing conditions can be optimized according to known methods. Based on these results, random DNA chain with modified A and T and unmodified C and G is evaluated for accurate detection and discrimination by the nanopore. These procedures allow characterization of the signals from each of the nucleotides and the transitions between nucleotides of different identities. The magnitude and duration of the blockade signatures on the event diagram are then analyzed and compared with known diagrams for validation. The schematic of the predicted blockade signals from DNA molecules (ii), (iii) and (iv) are shown in FIG. 7. Thus, with these rational chemical designs and modifications of the building blocks of DNA, this invention envisions using nanopore to decipher DNA sequences at the single molecule level with single base resolution.

Attach Small Hooks to the Nucleotides for Synthesis of DNA in Polymerase Reaction for Nanopore Detection

If a DNA polymerase is not able to synthesize a long strand of DNA due to the bulkiness of the functional groups introduced, an alternative strategy is to introduce small ‘hooks’ to the nucleotides, then perform polymerase reaction to produce DNA products with hook-labeled nucleotides incorporated in them.

The DNA products are then linked with the large functional groups through the hook for distinct detection by nanopore.

(1) Design and Synthesis of Hook-Labeled Nucleotide duTP-N3.

The available dCTP, dGTP and dATP-NH2 are used as starting materials directly (FIG. 8), while dUTP-N3 is synthesized from 5-iodo-2′-deoxyuridine as shown in FIG. 9. 5-Iodo-2′-deoxyuridine is first coupled with propargylamine in the presence of palladium(0) and copper(I) catalysts. Then the amino group is converted into azido group by the diazo transfer method [11]. Finally triphosphate is introduced to the 5′-hydroxy group of the nucleoside to yield dUTP-N3 [6].

(2) DNA-Extension Reaction Using Hook-Labeled Nucleotides (dATP-NH2 and dUTP-N3).

The dATP-NH2 and dUTP-N3, and the unmodified dCTP and dGTP, are used in polymerase reaction on the single-stranded nucleic acid template to obtain hook-labeled DNA products. Due to the small sizes of the azido and amino groups, these nucleotides are expected to be good substrates of commonly used DNA polymerases. After isolation of the single stranded DNA carrying the hook, the azido groups on these modified DNA chains will be further modified by Huisgen 1,3-dipolar cycloaddition with terminal alkynes (R3C≡CH) in the presence of copper(I) catalyst (FIG. 10) [18 and 19]. The amino groups on the “A” nucleotides of these modified DNA chains are connected with the modification groups R1 in DMF and NaHCO3/Na2CO3 buffer solution [13 and 17]. After modification, the order of nucleotides bulkiness on the chain will be: A*>U*>G>C since in general the modification group R1 is larger than R3.

Nanopore Contruction and Detection of DNA

Based on information in the art, nanopores are constructed with different configurations and modifications for characterizing DNA containing nucleotides of different sizes.

Synthetic nanopores are described in references [24] through [28] which are hereby incorporated by reference in their entirety. The mechanics and kinetics of DNA passage through the pores are described in references [29] and [30], respectively.

Natural nanopores are described in references [31] through [34] which are hereby incorporated by reference in their entirety.

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Classifications
Classification aux États-Unis435/6.1, 536/23.1
Classification internationaleC12Q1/68, C07H21/04
Classification coopérativeC12Q1/6869, C12Q2525/101, C12Q2565/631
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